Serine beta-lactamases are the major resistance mechanism to beta-lactam antibiotics, such as penicillin. In the last 20 years, hundreds of mutant beta-lactamases have appeared in response to an ever-growing number of plactams. These enzymes pose a pressing medical problem and a fascinating example of molecular evolution happening as we watch. A goal of this research is to understand the molecular bases of this evolving activity. A second goal is to exploit this information to discover novel beta-lactamase inhibitors, which may escape the current cycle of incremental antibiotic modification followed by rapid resistance response. The specific aims are: 1. To understand the molecular bases of action of evolved mutant a-lactamases. Outstanding questions include: How do "extended spectrum" substitutions increase activity against "beta -lactamase-stable" beta-lactams, such as ceftazidime? Correspondingly, how do "inhibitor resistant" substitutions confer resistance to inhibitors such as clavulanate? Similar to many resistance enzymes, these substitutions often occur far from the active site-how do they affect activity? Does the increase in activity have a cost? How, for instance, is new activity gained without activity loss against traditional substrates? Correspondingly, do these mutants, which often involve significant re-arrangements of the active site, lose stability? We target mutants of the TEM, CTX-M, and AmpC families, which wide-spread. Characteristic mutants will be cloned, over-expressed, their kinetics measured and their structures determined by x-ray crystallography. Using substrate and transition-state analogs, both apo- and ligand-bound structures will be determined. 2. To discover novel inhibitors of wild-type and mutant a-lactamases. Both novel inhibitors, not resembling beta-lactams, and potent substrate and transition-state analogs, will be designed and tested as agents to reverse a-lactamase-based resistance. Guided by the structural studies from aim 1, the following questions will be explored: Can non-beta-lactams inhibit beta-lactamases potently? Can we imagine an "uber" inhibitor that will target both WT and the diverse classes of mutant enzymes, or will a more specialist strategy be required? Will novel inhibitors evade the pre-evolved resistance mechanisms that face beta-lactams? Four classes of inhibitors will be synthesized and compared: substrate analogs, transition-state analogs, arylboronic acids, and leads from virtual screening. The Kj values of the new inhibitors and their crystal structures with mutant and WT beta-lactamases will be determined. The antimicrobial synergy of the new inhibitors will be evaluated.